Precursor used for labeling hepatorcyte receptor and containing trisaccharide and diamide demercaptide ligand, method for preparing the same, radiotracer and pharmaceutical composition of the same

ABSTRACT

A precursor used for labeling hepatocyte receptors and applied to radiotracers for imaging or pharmaceutical compositions for liver cancers is revealed. The precursor is a bifunctional compound. The bifunctional group includes a trisaccharide structure and a diamide dimercaptide (N 2 S 2 ) ligand. The trisaccharide has high affinity to asialoglycoprotein receptors (ASGPR) on surfaces of hepatocytes while N 2 S 2  ligand reacts with radioisotopes to form neutral complexes. Thus the precursor stays on surfaces of hepatocytes to provide radioisotope labeling or treatment effect of liver cancers.

BACKGROUND OF THE INVENTION

1. Fields of the invention

The present invention relates to a precursor used for labelinghepatocyte receptors, a method for preparing the same, a radiotracersfor imaging, and a pharmaceutical composition of the same, especially toa bifuncitonal precursor containing trisaccharide and a diamidedimercaptide (N₂S₂) ligand, a method for preparing the same, aradiotracer, and a pharmaceutical composition of the same.

2. Descriptions of Related Art

Faced with an increasingly ageing and more competitive society, variouskinds of diseases such as cancers, cerebrovascular diseases, diseases ofnervous system and heart diseases seriously threaten our health. Forearly diagnosis, early treatment and prevention of these diseases, thedevelopment of functional and molecular techniques of diagnostic andtreatment of the above diseases have become matters of great urgency.

In medical techniques available now, isotope tracer techniques and serumbiochemical markers are commonly used for detecting human diseases orfunctional disorders. Both have advantages of safety, non-invasion,convenience and accuracy. As to the treatment target, the most popularapplications are related to follow-up and treatment of liver diseases.Among liver diseases, liver fibrosis represents the liver's response tosome simulates such as necrosis and inflammation and these stimulatesarouse durative hyperplasia of fibril connective tissue in the liver.Liver fibrosis is reversible in early stage. The advanced liver fibrosisresults in cirrhosis and cirrhosis is generally irreversible.

The main method for testing and diagnosis of liver fibrosis is liverpuncture to get liver biopsy. The method has following shortcomings.Firstly, this is an invasive test method. Secondly, the method has therisk of complications including pain, bleeding, peritonitis, etc. Thethird, if the liver tissue obtained is too small or too short, there isan error in test results and clinical diagnosis. The fourth is that thetest is with low reproducibility. The test is unable to be applied topatients on clinical repetitively for monitoring patient's conditionsdynamically

According to international standard, liver fibrosis includes fourstages. In the F1/F2(mild/moderate) stages, liver fibrosis is reversibleand the treatment effect is optimal. The liver tissue can be back tonormal state. In the F 3 stage (advanced fibrosis), the treatment effectis poor and liver fibrosis is severe. As to the stage F4, liver fibrosisis worsen to liver cirrhosis and is irreversible. Thus the earlier liverfibrosis is detected, the better the treatment effect of the patient.

Human cells have specific receptors on surfaces to accept some specificproteins or peptides. According to this specificity, some proteins orpeptides are labeled with radioactive nuclides and are delivered intohuman bodies. Then the labeled proteins or peptides achieve higherconcentration in specific organs or tissues so as to diagnose or treatdiseases by using nuclear imaging.

In the past, bifunctional group ligand is used together with technetiumcompounds or rhenium compounds to label proteins or peptides. Forexample, compound S-Hynic with bifunctional group includes an activecarboxylic acid that is used to form a strong amide bond with proteinsor peptides. Moreover, it contains a pyridyl group and hydrazo structurethat bond to ^(99m)Tc. While being used together with auxiliarychelating agents such as Tricine, S-Hynic reacts with technetium orrhenium to get stable complexes. However, S-Hynic solution isphotosensitive and is not convenient in use. Thus there is a need tofind out more stable organic compounds with bifunctional groups.

There are about two hundred thousand asialoglycoprotein receptors(ASGPR) on surfaces of mammalian heptocytes. The asialoglycoproteinreceptor (ASGPR) is a liver-specific transmembrane glycoprotein thatmediates endocytosis, removes desialylated glycoproteins, and involvesin lipoprotein catabolism. The ASGPR also has high affinity to galactose(Gal) and N-acetylgalactosamine (GalNAc). Especially when a groundsubstance contains tri-Gals or N-acetylgalactosamine, it has higheraffinity to ASGPR on surfaces of hepatocytes, almost 10⁶ times than asubstrate with a single N-acetylgalactosamine. Based on thischaracteristic, YEE (ah-GalNAc); has been used as a drug/gene carrierfor drug or gene delivery to hepatocytes.

It is learned that carboxylic acids can bind to alcohols,saccharides(carbohydrates), amines, amino acids, peptides and proteinswhile diamide dimercaptide (N₂S₂) ligands bind to radioisotopes such astechnetium or rhenium. But now there is no radioactive tracer forimaging of hepatocyte receptors formed by Gal, GalNAc and N₂S₂ ligand.

Both Gal and GalNAc have specificity to hepatic lectin. Onceradioisotopes are connected to Gal and GalNAc of glycoprotein, nuclearpharmaceuticals are optimally delivered to the targeted liver cells andentered the cells by endocytosis for functional imaging or therapeuticuse. The design of glycosyl group is not revealed yet in the field ofnuclear medicine. Thus a preparation method for hepatocyte receptorlabeled precursor containing trisaccharide and a diamide dimercaptide(N₂S₂) ligand is provided so as to increase sensitivity and specificityof nuclear medicine tests.

SUMMARY OF THE INVENTION

Therefore it is a primary object of the present invention to provide aprecursor used for labeling hepatocyte receptors and containingtrisaccharide and a diamide dimercaptide (N₂S₂) ligand, a method forpreparing the same, a radiotracer, and a pharmaceutical composition ofthe same. The N₂S₂ ligand is used to chelate radioisotopes and the threeactive carboxylate ester that reacts with compounds containing aminogroups to form amide bonds. Thus a bifunctional compound is obtained.The bifunctional compound can bond to polyols, saccharides, amines,amino acids, peptides or proteins so as to stay on surfaces ofhepatocytes. On the other hand, the compound can also bond to othercompounds such as TcO³⁺, ReO³⁺, etc. and then is applied to research andproduction of nuclear pharmaceuticals.

It is another object of the present invention to provide a precursorused for labeling hepatocyte receptors and containing trisaccharide anda diamide dimercaptide (N₂S₂) ligand, a method for preparing the same, aradiotracer, and a pharmaceutical composition of the same in whichtrityl groups are used to protect thiol groups of N₂S₂ ligand. Duringthe complex reaction, the protecting groups are released automaticallyand there is no need to remove the protecting groups in advance beforethe complex reaction. Thus this is more convenient to use.

It is a further object of the present invention to provide a precursorused for labeling hepatocyte receptors and containing trisaccharide anda diamide dimercaptide (N₂S₂) ligand, a method for preparing the same, aradiotracer, and a pharmaceutical composition of the same in whichgalactoside ah-GalNAc₄ is used as a functional group and the galactosidehas high affinity to asialoglycoprotein receptors (ASGPR) on surfaces ofheptocytes so as to ensure imaging effect of liver and treatment effectof liver cancers.

It is a further object of the present invention to provide a precursorused for labeling hepatocyte receptors and containing trisaccharide anda diamide dimercaptide (N₂S₂) ligand, a method for preparing the same, aradiotracer, and a pharmaceutical composition of the same. During thesynthesis of galactoside ah-GalNAc₄, benzyloxycarbonyl (carboxybenzyl,cbz) is an amine protecting group used to protect an amino group of6-aminohexanol. Thus trifluoroacetyl compounds are no more used. Thisprotecting group is easily to be released during hydrogenation.Moreover, other functional groups of the molecule are not affected bythis protecting group during hydrogenation.

In order to achieve the above objects, a precursor used for labelinghepatocyte receptors and containing trisaccharide and a diamidedimercaptide (N₂S₂) ligand, a method for preparing the same, aradiotracer, and a pharmaceutical compositions of the same are provided.The method for preparing the precursor includes a plurality of steps.Firstly synthesize a carboxylic acid of a bifunctional chelating agentcontaining a diamide dimercaptide (N₂S₂) ligand. Then the carboxylicacid is amidated to form an amide of the bifunctional chelating agentcontaining N₂S₂ ligand and hydrolyze the amide to get three polycarboxylgroups. Next synthesize three galactosides. At last, use thegalactosides to amidate the polycarboxyl groups of the amide and get aprecursor used for labeling hepatocyte receptors and containingtrisaccharide and a N₂S₂ ligand. Moreover, the precursor can be used toprepare radiotracers for imaging and form a pharmaceutical compositionfor treating liver cancers.

BRIEF DESCRIPTION OF THE DRAWINGS

The structure and the technical means adopted by the present inventionto achieve the above and other objects can be best understood byreferring to the following detailed description of the preferredembodiments and the accompanying drawings, wherein

FIG. 1 shows a chemical structure of a precursor used for labelinghepatocyte receptors and containing trisaccharide and a diamidedimercaptide (N₂S₂) ligand according to the present invention;

FIG. 2 is a flow chart showing steps of preparing precursor used forlabeling hepatocyte receptors and containing trisaccharide and a diamidedimercaptide (N₂S₂) ligand according to the present invention;

FIG. 3 shows a part of flow chart of synthesis of an embodimentaccording to the present invention;

FIG. 4 shows a part of flow chart of synthesis of an embodimentaccording to the present invention;

FIG. 5 shows a part of flow chart of synthesis of an embodimentaccording to the present invention;

FIG. 6 shows a part of flow chart of synthesis of an embodimentaccording to the present invention;

FIG. 7 shows a part of flow chart of synthesis of an embodimentaccording to the present invention;

FIG. 8 shows a chemical structure a radiotracer or a pharmaceuticalcomposition according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Conventional labeling of human liver cells has shortcomings of poorlabeling effect, low stability and inconvenience in use. The presentinvention provides a compound with a specific chemical structure and amethod for preparing the same that overcome the above problems.

Refer to FIG. 1, it is a schematic drawing showing a chemical structureof a precursor used for labeling hepatocyte receptors and containingtri-galactose and a diamide dimercaptide (N₂S₂) ligand of the presentinvention. Without chelating radioisotopes, the N₂S, ligand is protectedby a trityl group because thiols are easy to be oxidized. Before acomplex reaction occurs between the N₂S₂ ligand and radioisotopes toproduce neutral complexes, a protecting group for thiol groups needs tobe removed. The trityl group used in the present invention is releasedduring the complex reaction and there is no need to remove the tritylgroup in advance. Thus the trityl group is convenient in use.

Beside the N₂S₂ ligand, the present invention further includestrisaccharide that has high affinity and specificity to ASGPR and entershuman HepG2 selectively by endocytosis. In other words, thistrisaccharide structure helps the precursor of the present invention tobe targeted to liver tumor cells. Then the precursor is delivered intoliver tumor cells by endocytosis so as to achieve radioactive labelingor therapeutic use.

Refer to FIG. 2, a flow chart of a method for preparing the precursorused for labeling hepatocyte receptors and containing tri-galactose andN₂S, ligand is revealed. The method includes following steps.

-   Step S1: Synthesize a carboxylic acid of a bifunctional chelating    agent containing a diamide dimercaptide (N₂S₂) ligand;-   Step S2: Amidate the carboxylic acid to form an amide of the    bifunctional chelating agent containing the diamide dimercaptide    (N₂S₂) ligand; further hydrolysis the amide to form three    polycarboxyl groups;-   Step S3: Synthesize three galactosides;-   Step S4: Use the galactosides to amidate the polycarboxyl groups of    the amide and get a precursor used for labeling hepatocyte receptors    and containing trisaccharide and a N₂S₂ ligand.

In these steps, the key technique features on the construction of thediamide dimercaptide (N₂S₂) ligand and trisaccharide structure. Thatmeans to synthesize a bifunctional compound that chelates radioactiveisotopes and has high affinity to ASGPR.

In the step S1, the carboxylic acid of a bifunctional chelating agentcontaining a diamide dimercaptide (N₂S₂) ligand used is7,10-diaza-9-oxo-7-[2-((triphenylmethyl)thio)-ethyl]-12-[(triphenylmethyl)thio]dodecanoicacid. The detailed steps for synthesis of this carboxylic acid are shownin the FIG. 3.

Step (1): For protecting thiol groups, take 2-thioethylaminehydrochloride and triphenylmethanol as reactants and borontrifluoride-diethyl ether complex is used as a catalyst to get2-[(triphenylmethyl)thio]ethylamine. The reaction temperature is 72degrees Celsius and the reaction time is 4 hours.Step (2): Use 2-[(Triphenylmethyl)thio]ethylamine and chloroacetylchloride to carry out the amidation reaction in trichloromethanesolution so as to produceN-[2-((Triphenylmethyl)thio)ethyl]-chloroacetamide.Step (3): Take 2-[(Triphenylmethyl)thio]ethylamine,N-[2-((Triphenylmethyl)thio)ethyl]-chloroacetamide and use triethylamineused as a reagent to carry out the substitution reaction indichloromethane solution. Thus an amine-amide-thiol ligand is produced.The reaction temperature of the substitution reaction is 55° C. and thereaction time is 48 hours.Step (4): Dissolve 6-bromohexanoic acid and thionyl chloride in absolutemethanol solution to carry out the esterification reaction and getmethyl 6-bromohexanoate. The reaction temperature of the esterificationreaction is 25° C. and the reaction time is 24 hours.Step (5): Take the amine-amide-thiol ligand and methyl 6-bromohexanoateand use sodium hydroxide (NaOH) as a reagent to carry out thesubstitution reaction in acetonitrile solution and get methyl7,10-diaza-9-oxo-7-[2-((triphenylmethyl)thio)-ethyl]-12-[(triphenylmethyl)thio]dodecanoate.The reaction temperature of the substitution reaction is 85° C. and thereaction time is 24 hours.Step (6): Hydrolyze methyl7,10-diaza-9-oxo-7-[2-((triphenylmethyl)thio)-ethyl]-12-[(triphenylmethyl)thio]dodecanoatein alkaline methanol solution to get7,10-diaza-9-oxo-7-[2-((triphenylmethyl)thio)-ethyl]-12-[(triphenyl-methyl)thio]dodecanoicacid.

After getting this carboxylic acid, take the step S2 to form amide byamidation of the carboxylic acid and gettrimethyl-γ-L-glutamyl-L-glutamyl-7,10-diaza-9-oxo-7-[2-(triphenylmethyl)thioethyl]-12-[(triphenylmethyl)-thio]dodecanamide.The detailed steps are shown in the FIG. 4. The steps are as followings.

Step (7) Dissolve γ-L-glutamyl-L-glutamic acid and thionyl chloride inabsolute methanol solution to carry out the esterification reaction andget methyl γ-L-glutamyl-L-glutamate; andStep (8) Take methyl γ-L-glutamyl-L-glutamate and the carboxylic acidand use 1,3-dicyclohexylcarbodiimide as well as N-hydroxysuccinimide asa reagent to carry out the amidation reaction in chloroform solution andgettrimethyl-γ-L-glutamyl-L-glutamyl-7,10-diaza-9-oxo-7-[2-(triphenylmethyl)thioethyl]-12-[(triphenylmethyl)-thio]dodecanamide.

Refer to FIG. 5,trimethyl-γ-L-glutamyl-L-glutamyl-7,10-diaza-9-oxo-7-[2-(triphenylmethyl)thioethyl]-12-[(triphenylmethyl)-thio]dodecanamideis hydrolyzed to get compound DODGA with three polycarboxylic groups.The polycarboxylic groups in the present invention are used to bond thegalactosides.

After completing the step S2, run the step S3 to synthesize compoundah-GalNAc₄ containing three galactosides. The galatoside is6′-aminohexyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactoside(ah-GalNAc₄). Refer to FIG. 6, it shows detailed steps for preparing theabove galatoside.

Step (9): Use 6′-aminohexanol and benzyl chlorocarbonate to carry out anamino protecting reaction and get a6′-(N-Benzyloxycarbonyl)aminohexanol.Step (10): Take N-acetyl-D-galactosamine and acetyl chloride to react at10° C. and get2-Acetamido-2,4,6,-tri-O-acetyl-1-chloro-1,2-dideoxy-α-D-galactopyranose.Step (11): Use 6′-(N-Benzyloxycarbonyl)aminohexanol and2-Acetamido-2,4,6,-tri-O-acetyl-1-chloro-1,2-dideoxy-α-D-Galactopyranoseto perform substitution reaction in a mixture solution of toluene andnitromethane with a catalyst of mercuric cyanide and get6′-(N-Benzyloxycarbonyl)aminohexyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranoside.Step (12) Hydrogenize/reduce 6′-(N-Benzyloxycarbonyl)aminohexyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosidein alcohol and in the presence of a palladium carbon catalyst to get the6′-aminohexyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactoside.

During the synthesis process of the above galactoside, a plurality ofprotecting groups can be used. Which protecting group is optimal dependson the use of the compound. For convenience of the following procedures,carboxybenzyl is used as the protecting group. Carboxybenzyl is easy tobe released and other functional groups of the molecule are not affectedby carboxybenzyl during hydrogenation process.

6′-(N-Benzyloxycarbonyl)aminohexyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosideobtained in the step S11 is separated and purified by liquidchromatography. Generally, a Sephadex LH-20 column is used for liquidchromatography. However, the Sephadex LH-20 column is quite expensive.The present invention uses silica gel to replace Sephadex LH-20 media.The purification effect is good and the cost is significantly reduced.

The galactoside ah-GalNAc₄ obtained by hydrogenation/reduction in thestep S12 doesn't need to be separated and purified. If a semi-product ofthe galactoside ah-GalNAc₄ before hydrogenation is with high purity,only toluene is produced during the hydrogenation process. The tolueneis extremely volatile so that it will evaporate completely withoutresidues during concentration processes. If a semi-product of thegalactoside ah-GalNAc₄ before hydrogenation is not of sufficient purity,the product obtained after hydrogenation needs to be separated andpurified. The process is similar to that of the step S11, a silica gelcolumn for liquid chromatography is used for separation andpurification.

The galactoside ah-GalNAc₄ obtained is used to connect with the compoundDODGA with three polycarboxylic groups previously prepared so as to form(ah-GalNAc)₃-DODGA that is a precursor used for labeling hepatocytereceptors and containing trisaccharide and a diamide dimercaptide (N₂S₂)ligand according to the present invention. Refer to FIG. 7, the stepsare as followings.

Step (13): Activate the carboxylic acid of DODGA to react withah-GalNAc₄ and use 1,3-dicyclohexylcarbodiimide as well asN-hydroxysuccinimide as a reagent to carry out the amidation reaction inchloroform solution and get6-Tri-o-(2′-acetamido-3′,4′,6′-tri-o-acetyl-2′-deoxy-β-D-galactopyranoside)hexyl7,10-diaza-9-oxo-7-[2-(triphenylmethyl)thioethyl]-12-[(triphenylmethyl)thio]-dodecanamido-γ-L-glutamyl-L-glutamate;andStep (14): Hydrolyze6-Tri-o-(2′-acetamido-3′,4′,6′-tri-o-acetyl-2′-deoxy-β-D-galactopyranoside)hexyl7,10-diaza-9-oxo-7-[2-(triphenylmethyl)thioethyl]-12-[(triphenylmethyl)thio]-dodecanamido-γ-L-glutamyl-L-glutamatewith sodium methylate to get6-Tri-o-(2′-acetamido-3′,4′,6′-trihydroxy-2′-deoxy-β-D-galactopyranoside)hexyl7,10-diaza-9-oxo-7-[2-(triphenylmethyl)thioethyl]-12-[(triphenylmethyl)thio]-dodecanamido-γ-L-glutamyl-L-glutamate,(ah-GalNAc)₃-DODGA.

While in use, radioactive isotope compound MO³⁺ is bound to diamidedimercaptide (N₂S₂) ligand to form a neutral complex. Now a trityl groupfor protecting thiol group in the N₂S₂ ligand is removed by dissolving(ah-GalNAc)₃-DODGA in trifluoroacetic acid and adding excess amount oftriethylsilane into the solution. Thus the trityl group is released fromthiol to form solid insoluble in trifluoroacetic acid. Users can removethe solid by filtering or wash the solid with n-Hexane. This isconvenient and simple.

Besides the precursor used for labeling hepatocyte receptor andcontaining trisaccharide and a diamide dimercaptide (N₂S₂) ligand andthe method for preparing the same, the present invention also reveals aradiotracer or a pharmaceutical composition of the same for treatment ofliver cancers. Refer to FIG. 8, a graph showing chemical structure ofradiotracers for imaging or pharmaceutical composition of the precursorused for labeling hepatocyte receptors and containing trisaccharide anddiamide dimercaptide (N₂S₂) ligand is disclosed. M is a radioactiveisotope such as ^(99m)Tc, ¹⁸⁸Re of ¹¹¹In that is used to label livercancer cells or treat liver cancer.

Beside N₂S₂ ligand used to chelate radioisotopes, the present inventionalso includes a trisaccharide structure. The trisaccharide structure hashigh affinity and specificity to ASGPR. Moreover, the trisaccharidestructure is selectively taken into human liver cancer cell line HepG2by endocytosis. Thus this structure helps the present invention to betargeted to liver cancer cells and enable the radioisotope to inhibit orkill the liver tumors. Therefore the pharmaceutical composition can beapplied to treat liver cancers. Furthermore, due to excellentperformance on the targeting, the precursor can be used as materials forradiotracers, used to label or treat liver cancer cells.

The compound of the present invention-(ah-GalNAc)₃-DODGA containsgalactoside-ah-GalNAc₄ and has high affinity to ASGPR on surfaces ofhepatocytes. Before use, there are some protecting groups for protectingthiol groups so that the compound is with high stability. Moreover,there is no need to remove the protecting groups in advance beforereacting with radioisotope to form complexes. Thus a radiotracer fordetecting liver fibrosis or a pharmaceutical composition for treatmentof liver cancers associated with the precursor of the present inventionis high-efficient and of significant medical values.

The followings are details and related parameters of each step accordingto the present invention.

Synthesis of 2-[(Triphenylmethyl)thio]ethylamine

Dissolve 2-thioethylamine hydrochloride (5 g, 44.2 mmol),triphenylmethanol (11 g, 42.5 mmol) and triethylamine (7 mL, 49.9 mmol)in 100 mL chloroform. Heat and reflux until the solution temperaturereaches a certain temperature, slowly drop a catalyst borontrifluorideethyl ether complex (15 mL, 119.5 mmol) into the solution and continueheating and reflux for 4 hours. Then cool down, add sodium bicarbonateaqueous solution into the solution and stir the mixture. White solidproduct is precipitated out immediately. Get the solid by suctionfiltration, wash the solid with water, and dry the solid. Thus solidproduct 2-[(triphenylmethyl)thio]ethylamine (14.0 g, 99%) is obtained.

Compound Data of the Product:

IR (neat) v 3381 (NH₂) cm⁻¹.

¹H NMR (CDCl₃) δ 7.42 (m, 3H, Ph), 7.30 (m, 12H, Ph), 2.58 (t, J=6.6 Hz,2H, CH₂N), 2.32 (t, J=6.6 Hz, 2H, CH₂S), 1.45 (br, 2H, NH₂).

¹³C NMR (CDCl₃) δ 144.80, 192.52, 127.81 and 126.60 (Ph), 66.51 (CPh),40.94 (CH₂N), 36.09 (CH₂S).

MS m/z 319 (M⁺), 243((CPh₃)⁺).

Synthesis of N-[2-((Triphenylmethyl)thio)ethyl]-chloroacetamide

Dissolve 2-[(triphenylmethyl)thio]ethylamine (5.24 g, 16.4 mmol) andtriethylamine (2.76 mL, 19.6 mmol) in 150 mL dichloromethane.Chloroacetyl chloride (1.56 mL, 19.6 mmol) is dissolved in 20 mLchloroform. Being cooled down with an ice bath, slowly drop chloroacetylchloride into the solution. Then stir the solution at room temperaturefor 2 hours. Next wash organic phase with followings in turn: 2Nhydrochloric acid solution (120 mL), and saturated sodium bicarbonateaqueous solution (100 mL). The organic phase is dehydrated by anhydroussodium sulfate (Na₂SO₄) and then is concentrated in vacuo to yieldyellow oily N-[2-((Triphenylmethyl)thio) ethyl]-chloroacetamide (5.62 g,86.6%).

Compound Data of the Product:

IR (neat) v 3413 and 3306 (NH), 1662 (CO) cm⁻¹.

¹H NMR (CDCl₃) δ 7.41 (m, 3H, Ph), 7.24 (m, 12H, Ph), 6.48 (br, 1H, NH),3.97 (s, 2H, CH₂Cl), 3.12 (q, J=6.3 Hz, 2H, CH₂N), 2.43 (t, J=6.3 Hz,2H, CH₂S).

¹³C NMR (CDCl₃) δ 165.63 (CO), 144.47, 129.48, 127.97 and 126.81 (Ph),66.52 (CPh), 42.54 (CH₂Cl), 38.35 (CH₂N), 31.67 (CH₂S).

MS m/z 397 and 395 (M⁺), 243 ((CPh₃)⁺).

Synthesis of[N-[2-((Triphenylmethyl)thio)ethyl][2-((triphenylmethyl)thio)ethyl-amino]acetamide]

Dissolve N-[2-((Triphenylmethyl)thio)ethyl]-chloroacetamide (5.4 g, 13.8mmol) and 2-[(triphenylmethyl)thio]ethylamine (4.4 g, 13.8 mmol) in 100mL dichloromethane, then add triethylamine (3.0 mL, 20.8 mmol) into thesolution, heat and reflux for 48 hours. After being cooled, wash with100 mL sodium bicarbonate aqueous solution (NaHCO₃) and take the organiclayer. After being dried with anhydrous sodium sulfate and concentrated,liquid chromatography (silicon dioxide, ethyl acetate hexane=1:1) isused for separation and purification so as to get light yellow oilproduct

N-[2-((Triphenylmethyl)thio)ethyl][2-((triphenylmethyl)thio)ethyl-amino]acetamide(2.2 g, 41.8%). Compound Data of the Product:

IR (neat) v 3330 (NH), 1670 (CO) cm⁻¹.

¹H NMR (CDCl₃) δ 7.42 (m, 4H, HNCO and Ph), 7.20 (m, 30H, Ph), 3.07 (m,4H, CH₂NCO and CH₂CO), 2.38 (m, 6H, CH ₂NHCH₂CO and CH₂S), 1.94 (br, 1H,NHCH₂CO).

¹³C NMR (CDCl₃) δ 170.84 (CO), 144.61, 129.47, 127.88 and 126.69 (Ph),66.72 and 66.65 (CPh₃), 51.62 (CH₂CO), 48.19 (CH₂NHCH₂CO), 37.70(CH₂NHCO), 32.12 and 31.97 (CH₂S).

MS m/z 243 ((CPH₃)⁺)

Synthesis of Methyl 6-bromohexanoate

Add 6-bromohexanoic acid (4.1 g, 21.1 mmol) into 100 mL absolutemethanol solution and then slowly drop 30 mL thionyl chloride into thesolution with an ice bath. Stir the solution at room temperatureovernight, concentrate the solution and add chloroform to dissolve.After suction filtration, take and concentrate the filtrate to get theproduct methyl 6-bromohexanoate (4.4 g, 100%).

Compound Data of the Product:

IR (neat) v 11739 (CO) cm⁻¹.

¹H NMR (CDCl₃) δ 3.64 (s, 3H, OCH ₃), 3.38 (t, 2H, BrCH ₂), 2.30 (t, 2H,CH ₂COOCH₃), 1.82 (m, 2H, CH₂CH ₂CH₂CH₂CH₂), 1.63 (m, 2H, CH₂CH₂CH₂CH₂CH₂), 1.46 (m, 2H, CH₂CH₂CH ₂CH₂CH₂).

¹³C NMR (CDCl₃) δ 173.77 (CO), 51.73 (COOCH₃), 33.72 (BrCH₂), 33.34(CH₂CH₂CH₂CH₂ CH₂), 32.30 (CH₂ CH₂ CH₂CH₂CH₂), 27.56 (CH₂CH₂CH₂ CH₂CH₂),23.98 (CH₂CH₂ CH₂CH₂CH₂).

Synthesis of Methyl7,10-diaza-9-oxo-7-[2-((triphenylmethyl)thio)-ethyl]-12-[(triphenylmethyl)thio]dodecanoate

TakeN-[2-((Triphenylmethyl)thio)ethyl][2-((triphenylmethyl)thio)ethyl-amino]acetamide(14.2, 21.0 mmol), add with methyl 6-bromohexanoate (17.6 g, 12.6 mmol),sodium hydroxide (1.76 g, 31.4 mmol), and 100 mL acetonitrile solution,heat and reflux overnight. After being cooled down and suction filtered,take the filtrate and concentrate the filtrate in vacuo. The residue isdissolved in 100 mL dichloromethane, washed with 100 mL water and removewater phase. The organic phase is dehydrated by anhydrous sodiumsulfate, concentrated and then purified by liquid chromatography(silicon dioxide, ethyl acetate:hexane=1:1) so as to get Methyl7,10-diaza-9-oxo-7-[2-((triphenylmethyl)thio)-ethyl]-12-[(triphenylmethyl)thio]dodecanoate(7.4 g, 44%).

Compound Data of the Product:

IR (neat) v 2928 (NH), 1735 and 1674 (CO) cm⁻¹.

¹H NMR (CDCl₃) δ 7.40 (NH), 7.30 (m, 30H, Ph), 3.64 (s, 3H, OCH₃), 3.02(q, 2H, NHCH ₂CH₂S), 2.83 (s, 2H, CO CH₂N), 2.36 (m, 4H, NHCH₂CH ₂S andNCH ₂CH₂S), 2.24 (m, 6H, NCH₂CH ₂S and CH ₂CH₂CH₂CH₂CH ₂), 1.53 (m, 2H,NCH₂CH ₂CH₂CH₂CH₂), 1.29 (m, 4H, CH₂CH₂CH ₂CH ₂CH₂).

¹³C NMR (CDCl₃) δ 173.90 and 171.13 (CO), 144.68, 144.70, 129.51,127.86, 126.66, (Ph), 77.43, 77.10 and 76.58 (CPh₃), 66.74 (CH₂N), 58.23(CH₃O), 54.54 and 53.82 (CH₂S), 51.40 (NHCH₂), 37.89 (NCH₂CH₂S), 33.89(CH₂CH₂CH₂CH₂CH₂), 31.97 (CH₂CH₂CH₂CH₂ CH₂), 29.94 (CH₂ CH₂ CH₂CH₂CH₂),26.72 (CH₂CH₂CH₂ CH₂CH₂), 24.67 (CH₂CH₂ CH₂CH₂CH₂).

Synthesis of7,10-Diaza-9-oxo-7-[2-((triphenylmethyl)thio)-ethyl]-12-[(triphenyl-methyl)thio]dodecanoicacid

Dissolve 20 g potassium hydroxide in 200 mL absolute methanol solution,add Methyl7,10-diaza-9-oxo-7-[2-((triphenylmethyl)thio)-ethyl]-12-[(triphenylmethyl)thio]dodecanoate(7.4 g, 9.2 mmol) into the solution and stir the solution at roomtemperature overnight. After being concentrated in vacuo at roomtemperature, add 30 mL water and 30 mL methanol. Adjust pH value of thesolution to 7.0 by using concentrated hydrochloric acid. Then extract by50 mL dichloromethane twice (2×50 mL), remove water phase and takeorganic phase layer. The organic phase layer is dehydrated by anhydroussodium sulfate and concentrated in vacuo to get7,10-Diaza-9-oxo-7-[2-((triphenylmethyl)thio)-ethyl]-12-[(triphenyl-methyl)thio]dodecanoicacid (6.9 g, 94.8%).

Compound Data of the Product:

IR (neat) v 3210 (OH), 2926 (NH), 1679 (CO) cm⁻¹.

¹H NMR (CD₃OD) δ 7.30 (m, 30H, Ph), 3.07 (t, 2H, COCH ₂N), 2.84 (m, 2H,NHCH ₂CH₂S), 2.75 (m, 2H, NCH ₂CH₂S), 2.64 (m, 2H, NCH₂CH ₂S), 2.37 (t,2H, NHCH₂CH ₂S), 2.28 (t, 2H, NCH ₂CH₂CH₂CH₂CH₂), 1.55 (m, 2H, CH₂COOH), 1.44 (m, 2H, CH₂CH₂CH₂CH ₂CH₂), 1.24 (m, 2H, CH₂CH₂CH ₂CH₂CH₂).

¹³C NMR (CD₃OD) δ 176.94 and 165.29 (CO), 146.03, 145.45, 130.69, 129.28and 127.81, 128.99, 128.28, 127.90 (Ph), 68.83 and 67.89 (CPh₃), 56(COCH₂), 55.29 (NCH₂ CH₂S), 55.13 (NCH₂CH₂CH₂CH₂CH₂), 39.68 (NHCH₂),34.44 (NCH₂CH ₂S), 32.55 (NHCH₂ CH₂S), 26.81 (CH₂COOH), 25.29 (CH₂CH₂CH₂CH₂CH₂), 24.52 (CH₂CH₂ CH₂CH₂CH₂).

MS m/z 243 ((CPh₃)⁺).

Synthesis of Methty γ-L-Glutamyl-L-glutamate

Add γ-L-Glutamyl-L-glutamic acid (H-Glu(Glu-OH)—OH) (2.0 g, 7.3 mmol)into 50 mL absolute methanol solution and then slowly drop 20 mL thionylchloride into the solution with an ice bath. Stir the solution at roomtemperature overnight and concentrate the solution to get the productmethyl γ-L-Glutamyl-L-glutamate (2.3 g, 100%).

Compound Data of the Product:

IR (neat) v 3368 (NH₂), 2924 (NH), 1735 and 1657 (CO) cm⁻¹.

¹H NMR (CDCl₃) δ 6.9 (br, NH), 4.52 (m, 1H, NH₂CH), 3.67 (s, 3H, OCH₃),3.65 (s, 3H, OCH₃), 3.44 (s, 3H₂OCH₃), 3.40 (q, 1H, NHCH), 2.32 (q, 2H,CH₂CH ₂CONH), 2.00 (m, 4H, CH ₂CH₂CONH and CH ₂CH₂COOCH₃), 1.78 (m, 2H,CH₂CH ₂COOCH₃).

¹³C NMR (CDCl₃) δ175.88, 173.16 and 172.34 (CO), 53.62 (NHCH), 52.35(NH₂CH), 52.07 (CH₂CH ₂CONH), 51.71 (CH₂CH ₂COO), 51.60 (CH₂ CH₂CONH),51.50 (CH₂CH₂COO).

MS m/z 320 (M⁺-Cl).

Synthesis ofTrimethyl-γ-L-glutamyl-L-glutamyl-7,10-Diaza-9-oxo-7-[2-(triphenylmethyl)thioethyl]-12-[(triphenylmethyl)-thio]dodecanamide

Take7,10-diaza-9-oxo-7-[2-((triphenylmethyl)thio)-ethyl]-12-[(triphenyl-methyl)thio]dodecanoicacid (0.55 g, 0.69 mmol), add with methyl γ-L-Glutamyl-L-glutamate (0.22g, 0.69 mmol), triethylamine (0.24 mL, 1.7 mmol), N-hydroxysuccinimide(0.10 g, 0.83 mmol), 1,3-dicyclohexylcarbodiimide (0.2 g, 0.1 mmol), and50 mL chloroform. Stir the solution at room temperature for 48 hours,suction filter, take the filtrate and wash the filtrate with water. Theresidue is dissolved in 100 mL acetone, then filter by suction and takethe filtrate. The organic phase is dehydrated by anhydrous sodiumsulfate, concentrated and purified by liquid chromatography (silicondioxide, chloroform:methanol=95:5) so as to getTrimethyl-γ-L-glutamyl-L-glutamyl-7,10-Diaza-9-oxo-7-[2-(triphenylmethyl)thioethyl]-12-[(triphenylmethyl)-thio]dodecanamide(0.31 g, 42.4%).

Compound Data of the Product:

IR (neat) v 3308 and 2927 (NH), 1741 and 1659 (CO) cm⁻¹.

¹H NMR (CDCl₃) δ7.46 (br, 1H, NHCH₂CH₂S), 7.25 (m, 30H, Ph), δ 6.80 (br,1H, CH₂CH₂CH₂CH₂CH₂CONH), 6.50 (br, 1H, CH₂CH₂CONH), 4.57 (m, 2H,NHCHCOOH₃), 3.72 (s, 6H, OCH₃), 3.66 (s, 3H, OCH₃), 3.00 (q, 2H, NHCH ₂CH₂S), 2.83 (s, 2H, COCH₂N), 2.19 (m, 16H, NHCH₂CH ₂S, NCH ₂CH ₂S, NCH₂CH₂CH₂CH₂CH ₂ and CH ₂CH ₂CO), 1.56 (m, 2H, NCH₂CH ₂CH₂CH₂CH₂), 1.23(m, 4H, NCH₂CH₂CH ₂CH ₂CH₂).

¹³C NMR (CDCl₃) δ 173.19, 173.14, 172.39, 172.14, 172.07 and 171.28(CO), 144.71, 144.67, 129.51, 127.87 and 126.67 (Ph), 77.45, 77.03 and76.60 (CPh₃), 66.47 (COCH₂), 58.13 and 54.56 (NHCH), 53.81 (CH₂CH₂CONH), 52.45 (CH₂CH₂CO), 51.76 (CH₂ CH₂CO), 37.90 (NCH₂CH₂), 3 6.06(NHCH₂CH₂), 32.18 (NHCH₂ CH₂) 31.95 (NCH₂ CH₂S), 30.02 (NCH₂CH₂CH₂CH₂CH₂), 27.90 (NCH₂ CH₂CH₂CH₂CH₂), 27.09, 26.80 and 26.61 (OCH₃),251.15 (NCH₂CH₂CH₂CH₂ CH₂).

MS m/z 243 ((CPh₃)⁺).

Synthesis of7,10-Diaza-9-oxo-7-[2-(triphenylmethyl)-thioethyl]-12-[(triphenylmethyl)thio]-dodecanamide-γ-L-glutamyl-L-glutamicacid (DODGA)

Dissolve 2 g potassium hydroxide in 20 mL absolute methanol solution,addtrimethyl-γ-L-glutamyl-L-glutamyl-7,10-Diaza-9-oxo-7-[2-(triphenylmethyl)thioethyl]-12-[(triphenylmethyl)-thio]dodecanamide(0.15 g, 0.15 mmol) into the solution and stir the solution at roomtemperature for 30 min. Add 10 mL water into the solution and adjust pHvalue of the solution into 7.0 by using concentrated hydrochloric acid.Then extract by 20 mL dichloromethane twice (2×20 mL), remove waterphase and take organic phase layer. The organic phase layer isdehydrated by anhydrous sodium sulfate and concentrated in under reducedpressure to get DODGA (0.15 g, 100%).

Compound Data of the Product:

IR (neat) v 3310 (OH), 3008 and 2927 (NH), 1741 and 1659 (CO) cm⁻¹.

¹H NMR (CD₃OD) δ 7.22 (m, 30H, Ph), 4.42 (m, 2H, NHCH), 3.65 (s, 2H,COCH₂), 3.05 (t, 2H, NHCH ₂CH₂S), 2.86 (m, 2H, CH ₂CH₂CH₂CH₂CH₂), 2.77(m, 2H, NCH ₂CH₂S), 2.65 (m, 2H, NCH₂CH ₂S), 2.35 (m, 4H, NHCH₂CH ₂S andCH₂CH ₂CO), 2.22 (m, 6H, CH ₂CH₂CONH, CH ₂CH₂COOH and CH₂CH₂CH₂CH₂CH ₂),1.99 (m, 2H, CH₂CH ₂COOH), 1.58 (m, 2H, CH₂CH ₂CH₂CH₂CH₂), 1.45 (m,2CH₂CH₂CH₂CH ₂CH₂), 1.27 (m, 2H, CH₂CH₂CH ₂ CH₂CH₂).

¹H NMR (CD₃OD) δ 175.14, 174.72, 174.58, 173.83, 173.79 and 173.76 (CO),144.91, 144.33, 129.58, 128.19, 127.91, 127.18 and 126.81 (Ph), 78.38(CPh₃), 67.71 (CH), 66.77, (COCH₂), 54.03 (NHCH₂CH₂S), 51.85 (CH₂CH₂CH₂CH₂CH₂), 38.59 (NCH₂CH₂S), 34.99 (NCH₂ CH₂S), 31.42 (NHCH₂ CH₂S), 30.18(CH₂CH₂CO), 26.63 (CH₂ CH₂CO), 25.59 (CH₂ CH₂CH₂CH₂CH₂), 24.81 (CH₂CH₂CH₂ CH₂CH₂), 23.30 (CH₂CH₂ CH₂CH₂CH₂).

MS m/z 243 ((CPh₃)⁺).

Synthesis of 6′-(N-Benzyloxycarbonyl)aminohexanol

Dissolve 6′-aminohexanol (5.9 g, 50.0 mmol) in 20 mL water, add withsodium carbonate (3.2 g, 30.0 mmol) and set the solution in an ice bath.Dissolve benzyl chlorocarbonate (7.3 g, 50.0 mmol) in 20 mL diethylether and slowly drop this solution into the above solution. Then stirthe mixture at room temperature for 2 hours. Filter the mixture and washsolid with a little amount of diethyl ether. Remove the solvent from thesolid in a vacuum system to get 6′-(N-Benzyloxycarbonyl) aminohexanol(9.2 g, 73.2%).

Compound Data of the Product:

IR (neat) v 3382 and 1530 (NH), 3336 (OH), 1688 (CO) cm⁻¹.

¹H NMR (CDCl₃) δ 7.34 (m, 5H, Ph), 5.08 (s, 2H, PhCH₂), 4.90 (br, 1H,NH), 3.60 (t, J=6.5 Hz, 2H, CH ₂OH), 3.17 (q, J=6.6 Hz, 2H, NHCH ₂),1.93 (br, 1H, OH), 1.52 (m, 4H, CH ₂CH₂CH₂CH ₂CH₂O), 1.35 (m, 4H, CH ₂CH₂CH₂CH₂O).

¹³C NMR (CDCl₃) δ 156.45 (CO), 136.55, 128.42 and 127.99 (Ph), 66.51(CH₂OH), 62.52 (PhCH₂), 40.82 (NHCH₂), 32.45 (CH₂CH₂OH), 29.84 (NHCH₂CH₂), 26.28 (CH₂CH₂CH₂OH), 25.22 (CH₂CH₂CH₂CH₂OH). MS m/z 251 (M⁺).

Synthesis of2-Acetamido-2,4,6,-tri-O-acetyl-1-chloro-1,2-dideoxy-α-D-galactopyranose

Cool down 30 mL acetyl chloride to 0° C., add withN-acetyl-D-galactosamine (3.0 g, 13.6 mmol), seal the solution with acover, then set and stir the solution in a thermostatic chamber at 10°C. After 5 days, add 80 mL dichloromethane into the solution and mixevenly. Then add 160 mL ice water into the solution, mix evenly and waituntil the two phases separate. The organic phase is washed once by 50 mLsaturated sodium bicarbonate aqueous solution (1×50 mL) and then isdehydrated anhydrous sodium sulfate. After evaporation of the solvenetunder reduced pressure, sticky oil product2-Acetamido-2,4,6,-tri-O-acetyl-1-chloro-1,2-dideoxy-α-D-galactopyranose(2.45 g, 51%) is obtained.

Compound Data of the Product:

IR (neat) v 3289 and 1544 (NH), 1750 and 1666 (CO) cm⁻¹.

¹H NMR (CDCl₃) δ 6.28 (d, J=3.6 HZ, 1H, H₁), 5.94 (d, J=8.7 Hz, 1H, NH),5.46 (dd, J=3.2 and 1.4 Hz, 1H, H₄), 5.29 (dd, J=11.4 and 3.3 Hz, 1H,H₃), 4.79 (m, 1H, H₂), 4.48 (t, J=6.9 Hz, 1H, H₅), 4.19 (m, 2H, H₆),2.17 (s, 3H, CH₃), 2.10 (s, 3H, CH₃), 2.03 (s, 3H, CH₃), 2.01 (s, 3H,CH₃).

¹³C NMR (CDCl₃) δ 170.65, 170.48, 170.26 and 169.95 (CO), 94.97 (C₁),69.73 (C₅), 67.27 (C₄), 66.48 (C₃), 61.06 (C₂), 49.12 (C₆), 22.91,20.56, 20.52 and 20.49 (CH₃).

MS m/z 330 (M⁺-Cl)

Synthesis of6′-(N-Benzyloxycarbonyl)aminohexyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranoside

Dissolve 6′-(N-Benzyloxycarbonyl)aminohexanol (0.72 g, 2.86 mmol),2-Acetamido-2,4,6,-tri-O-acetyl-1-chloro-1,2-dideoxy-α-D-galactopyranose(1.05 g, 2.86 mmol), anhydrous calcium sulfate (0.3 g) and mercuriccyanide (0.88 g, 3.5 mmol) in a mixture of 15 mL toluene and 15 mLnitromethane. Stir the mixture solution at room temperature for 24 hoursand then filter the mixture solution. The filtrate is concentrated invacuo, then dissolve residue in 80 mL dichloromethane and wash with 50mL water twice (2×50 mL). The organic phase is dried by anhydrous sodiumsulfate, concentrated under reduced pressure, separated and purified byliquid chromatography (silicon dioxide, chloroform methanol=95:5). Thuscolorless solid product 6′-(N-Benzyloxycarbonyl)aminohexyl2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranoside (0.58 g,35%) is obtained.

Compound Data of the Product:

IR (KBr) v 3318 and 1543 (NH), 1748 and 1668 (CO) cm⁻¹.

¹H NMR (CDCl₃) δ 7.35 (m, 5H, Ph), 5.94 (d, J=8.4 Hz, NH), 5.33 (d,J=3.0 H2, H₄), 5.26 (dd, J=11.3 and 3.2 Hz, 1H, H₃), 5.11 (AB, J=12.3Hz, 2H, CH₂Ph), 4.90 (br, 1H, NH), 4.65 (d, J=8.4 Hz, 1H, H₁), 4.12 (m,2H, H₆), 4.02-3.81 (m, 3H, H₂, H₅, and OCH ₂CH₂), 3.48 (m, 1H, OCH₂CH₂), 3.21 (m, 2H, CH₂N), 2.13 (s, 3H, CH₃), 2.05 (s, 3H, CH₃), 2.0 (s,3H, CH₃), 1.94 (s 3H, CH₃), 1.51 (m, 4H, OCH₂CH ₂CH₂CH₂CH ₂), 1.34 (m,4H, OCH₂CH₂CH ₂CH ₂).

¹³C NMR (CDCl₃) δ 170.44 and 156.56 (CO), 136.64, 128.49, 128.04 and127.84 (Ph), 100.73 (C₁), 70.51, (C₅), 69.36 (C₄), 69.34 (C₃), 66.79(CH₂Ph), 66.53 (OCH₂CH₂), 61.44 (C₆), 51.54 (C₂), 40.55 (CH₂NH), 29.70(OCH₂ CH₂), 28.93 (NHCH₂ CH₂), 25.89 (OCH₂CH₂ CH₂), 25.07 (CH₂CH₂CH₂N),23.35 (CH₃CONH), 20.67 (CH₃COO).

¹H NMR (CD₃OD) δ 7.34 (m, 5H, Ph), 5.32 (d, J=3.3 Hz, 1H, H₄), 5.05 (m,3H, H₃ and CH₂Ph), 4.54 (d, J=8.4 Hz, 1H, H₁), 4.15-3.95 (m, 4H, H₂, H₅and H₆), 3.83 (m, 1H, OCH₂), 3.48 (m, 1H, OCH₂), 3.10 (t, J=6.9 Hz, 2H,CH₂N), 2.13 (s 3H, CH₃), 2.01 (s, 3H, CH₃), 1.94 (s, 3H, CH_(:3)), 1.91(s, 3H, CH₃), 1.51 (m, 4H, OCH₂CH ₂CH₂CH₂CH ₂), 1.34 (m, 4H, OCH₂CH₂CH₂CH ₂).

¹³C NMR (CD₃OD) δ 173.52, 172.13 and 171.11 (CO), 158.88, 138.55, 129.49and 128.96 and 128.74 (Ph), 102.66 (C₁), 72.17 (C₅), 71.76 (C₄), 70.70(C₃), 68.25 (CH₂Ph), 67.27 (OCH₂CH₂), 62.79 (C₆), 51.69 (C₂), 41.73(CH₂N), 30.89 (OCH₂ CH₂), 30.51 (NHCH₂ CH₂), 27.45 (OCH₂CH₂ CH₂), 26.68(CH₂CH₂CH₂N), 22.93 (CH₃), 20.65 (CH₃).

MS m/z 521 (M⁺-CH₃).

Synthesis of 6′-Aminohexyl2-acetamio-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranoside (ah-GalNAc₄)

Dissolve 6′-(N-Benzyloxycarbonyl)aminohexyl2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranoside (0.66 g,1.14 mmol) in 20 mL alcohol and add 10% palladium carbon catalyst (0.08g) into the solution. Set the solution in a reduction device and vibratethe solution in 50 psi hydrogen gas. After 15-24 hours, filter thesolution and dry the filtrate under reduced pressure to get ah-GalNAc₄(0.51 g, 100%).

Compound Data of the Product:

IR (neat) v 3256 and 3377 (NH₂), 1747 and 1657 (CO) cm⁻¹.

¹H NMR (CD₃OD) δ 5.33 (d, J=2.7 Hz, 1H, H₄), 5.05 (dd, J=11.4 and 3.3Hz, 1H, H₃), 4.55 (d, J=8.4 Hz, 0.1H, H₁), 4.18-3.97 (m, 4H, H₂, H₅ andH₆), 3.86 (m, 1H, OCH ₂), 3.52 (m, 1H, OCH ₂), 2.92 (t, J=7.5 Hz, 2H,CH₂N), 2.14 (s, 3H, CH₃), 2.02 (s, 3H, CH₃), 1.94 (s, 3H, CH₃), 1.93 (s,3H, CH₃), 1.46 (m, 4H, OCH₂CH ₂CH₂CH₂CH ₂), 1.42 (m, 4H, OCH₂CH₂CH ₂CH₂).

¹³C NMR (CD₃OD) δ 172.03, 171.97 and 171.61 (CO), 102.66 (C₁), 72.12(C₅), 71.76 (C₄), 70.64 (C₃), 68.17 (OCH₂), 67.72 (C₆), 51.52 (C₂),40.75 (CH₂N), 30.19 (OCH₂ CH₂), 28.36 (CH₂CH₉N), 27.02 (OCH₂CH₂ CH₂),26.35 (CH₂CH₂CH₂N), 22.95 (CH₃), 20.58 (CH₃).

MS m/z 446 (M⁺), 387 (M⁺-CH₃COO).

Synthesis of6-Tri-o-(2′-acetamido-3′,4′,6′-tri-o-acetyl-2′-deoxy-(3-D-galactopyranoside)hexyl7,10-diaza-9-oxo-7-[2-(triphenylmethyl)thioethyl]-12-[(triphenylmethyl)thio]-dodecanamido-γ-L-glutamyl-L-glutamate

Take and put DODGA (1.32 g, 1.31 mmol), ah-GalNAc₄ (1.21 g, 5.87 mmol),triethylamine (0.82 mL, 5.87 mmol), 1,3-dicyclohexylcarbodiimide (1.21g, 5.87 mmol), N-hydroxysuccinimide (0.68 g, 5.87 mmol), and 80 mLchloroform into a 250 mL round-bottom flask, heat to 65° C. and refluxfor 24 hours. Concentrate the solution, dissolve EA in solution,concentrate the solution again and use liquid chromatography (silicondioxide, chloroform methanol=95:5) for separation and purification so asto get a product6-Tri-o-(2′-acetamido-3′,4′,6′-tri-o-acetyl-2′-deoxy-β-D-galactopyranoside)hexyl7,10-diaza-9-oxo-7-[2-(triphenylmethyl)thioethyl]-12-[(triphenylmethyl)thio]-dodecanamido-γ-L-glutamyl-L-glutamate(1.12 g, 37%).

Compound Data of the Product:

IR (neat) v 3287 and 2929 and 1538 (NH), 1748 and 1658 (CO) cm⁻¹.

¹H NMR (CDCl₃) δ7.42 (NH), 7.40-7.15 (m, 12H, Ph), 6.8-6.4 (NH), 5.35(s, CHOAC), 5.30 (m, NHCHCO), 5.15 (m, OCHO), 4.67 (t, CH ₂OAC), 4.14(m, CHNHAC), 3.91 (m, CHCH₂OAC), 3.70 (m, CH₂CH ₂O), 3.45 (m, CH ₂CH₂O),3.20 (m, NHCH₂CH ₂S), 3.00 (q, NCH ₂CH₂S), 2.83 (s, COCH₂N), 2.37 (m,NHCH₂CH ₂S and NCH₂CH ₂S), 2.22 (CH ₂CH₂CO), 2.05 (m, OAC), 1.54 (m, CH₂CH ₂CH₂CH₂CH ₂CH ₂), 1.34 (m, CH₂CH₂CH ₂CH ₂CH₂CH₂).

MS m/z 2335 (M⁺), 2357 (M⁺Na)⁺

Synthesis of6-Tri-o-(2′-acetamido-3′,4′,6′-trihydroxy-2′-deoxy-β-D-galactopyranoside)hexyl7,10-diaza-9-oxo-7-[2-(triphenylmethyl)-thioethyl]-12-[(triphenylmethyl)thio)-dodecanamido-γ-L-glutamyl-L-glutamate]

Dissolve6-Tri-o-(2′-acetamido-3′,4′,6′-tri-o-acetyl-2′-deoxy-β-D-galactopyranoside)hexyl7,10-diaza-9-oxo-7-[2-(triphenylmethyl)thioethyl]-12-[(triphenylmethyl)thio]-dodecanamido-γ-L-glutamyl-L-glutamate(1.12 g, 0.48 mmol) in 20 mL absolute methanol solution. Then add sodiummethylate/methanol solution (0.5M, 0.93 mL) into the solution and stirthe mixture at room temperature for 15 min. Slowly drop 0.1Nhydrochloric acid solution into the mixture with an ice bath and adjustpH value of the mixture into 6. After being concentrated under reducedpressure, (ah-GalNAc)₃-DODGA (0.94 g, 100%) is obtained.

Compound Data of the Product:

IR (KBr) v 3400 (OH), 2930 and 1539 (NH), 1630 (CO) cm⁻¹.

¹H NMR (CD₃OD) δ7.32-7.17 (m, 12H, Ph), 7.46 (d, CHOH), 4.20 (m, CHCO),3.95 (m, CHCHOH), 3.81 (m, OCHO), 3.77 (m, COCH₂), 3.68 (d, CH₂OH), 3.53(m, CH₂CH ₂O), 3.41 (m, CH ₂CH₂O), 3.23 (m, NHCH ₂CH₂S), 2.95 (m, NCH₂CH₂S), 2.60 (t, NHCH ₂CH₂), 2.29 (m, CH₂CH ₂CO), 2.10 (m, CH ₂CH₂CO),1.90 (s, NHAc), 1.45 (m, CH ₂CH ₂CH₂CH₂CH ₂CH ₂), 1.26 (m, CH₂CH₂CH ₂CH₂CH₂CH₂).

MS m/z 1957 (M⁺), 1981 (M⁺Na)⁺.

Additional advantages and modifications will readily occur to thoseskilled in the art. Therefore, the invention in its broader aspects isnot limited to the specific details, and representative devices shownand described herein. Accordingly, various modifications may be madewithout departing from the spirit or scope of the general inventiveconcept as defined by the appended claims and their equivalents.

What is claimed is:
 1. A precursor used for labeling hepatocyte receptorand containing trisaccharide and a diamide dimercaptide (N₂S₂) ligand isrepresented by the following structural formula


2. A method for preparing a precursor used for labeling hepatocytereceptor and containing trisaccharide and a diamide dimercaptide (N₂S₂)ligand comprising the steps of: synthesizing a carboxylic acid of abifunctional chelating agent containing a diamide dimercaptide (N₂S₂)ligand; amidating the carboxylic acid to form an amide of thebifunctional chelating agent containing N₂S₂ ligand and furtherhydrolyzing the amide to get three polycarboxyl groups; synthesizingthree galactosides; and using the galactosides to amidate thepolycarboxyl groups of the amide and get a precursor used for labelinghepatocyte receptors and containing trisaccharide and a N₂S₂ ligand. 3.The method as claimed in claim 2, wherein the carboxylic acid is7,10-diaza-9-oxo-7-[2-((triphenylmethyl)thio)-ethyl]-12-[(triphenyl-methyl)thio]dodecanoicacid.
 4. The method as claimed in claim 3, wherein the step ofsynthesizing a carboxylic acid further includes the steps of: step (1)taking 2-thioethylamine hydrochloride and triphenylmethanol to reactunder catalysis of boron trifluoride-diethyl ether complex forprotecting thiol groups and get 2-[(triphenylmethyl)thio]ethylamine;step (2) using 2-[(Triphenylmethyl)thio]ethylamine and chloroacetylchloride to carry out the amidation reaction in trichloromethanesolution so as to produceN-[2-((Triphenylmethyl)thio)ethyl]-chloroacetamide; step (3) taking2-[(Triphenylmethyl)thio]ethylamine, andN-[2-((Triphenylmethyl)thio)ethyl]-chloroacetamide and usingtriethylamine used as a reagent to carry out the substitution reactionin dichloromethane solution and get an amine-amide-thiol ligand; step(4) dissolving 6-bromohexanoic acid and thionyl chloride in absolutemethanol solution to carry out the esterification reaction and getmethyl 6-bromohexanoate; step (5) taking the amine-amide-thiol ligandand methyl 6-bromohexanoate and using sodium hydroxide (NaOH) as areagent to carry out the substitution reaction in acetonitrile solutionand get methyl7,10-diaza-9-oxo-7-[2-((triphenylmethyl)thio)-ethyl]-12-[(triphenylmethyl)thio]dodecanoate;step (6) hydrolyzing methyl7,10-diaza-9-oxo-7-[2-((triphenylmethyl)thio)-ethyl]-12-[(triphenylmethyl)thio]dodecanoatein alkaline methanol solution to get7,10-diaza-9-oxo-7-[2-((triphenylmethyl)thio)-ethyl]-12-[(triphenyl-methyl)thio]dodecanoicacid.
 5. The method as claimed in claim 2, wherein the carboxylic acidof the bifunctional chelating agent containing the diamide dimercaptide(N₂S₂) ligand uses a trityl group as a protecting group.
 6. The methodas claimed in claim 4, wherein in the step (1), reaction temperature ofthe reaction for protecting thiol groups is 72 degrees Celsius andreaction time is 4 hours.
 7. The method as claimed in claim 4, whereinin the step (3), reaction temperature of the substitution reaction is55° C. and reaction time is 48 hours.
 8. The method as claimed in claim4, wherein in the step (4), reaction temperature of the esterificationreaction is 25° C. and the reaction time is 24 hours.
 9. The method asclaimed in claim 4, wherein in the step (5), reaction temperature of thesubstitution reaction is 85° C. and the reaction time is 24 hours. 10.The method as claimed in claim 2, wherein the amide istrimethyl-γ-L-glutamyl-L-glutamyl-7,10-Diaza-9-oxo-7-[2-(triphenylmethyl)thioethyl]-12-[(triphenylmethyl)-thio]dodecanamide.11. The method as claimed in claim 10, wherein the step of amidating thecarboxylic acid further includes the steps of: step (7) dissolvingγ-L-glutamyl-L-glutamic acid and thionyl chloride in absolute methanolsolution to carry out the esterification reaction and get methylγ-L-glutamyl-L-glutamate, step (8) taking methylγ-L-glutamyl-L-glutamate and the carboxylic acid and using1,3-dicyclohexylcarbodiimide as well as N-hydroxysuccinimide as areagent to carry out the amidation reaction in chloroform solution andgettrimethyl-γ-L-glutamyl-L-glutamyl-7,10-diaza-9-oxo-7-[2-(triphenylmethyl)thioethyl]-12-[(triphenylmethyl)-thio]dodecanamide.12. The method as claimed in claim 11, wherein in the step (7), reactiontemperature of the esterification reaction is 25 degrees Celsius andreaction time is 24 hours.
 13. The method as claimed in claim 11,wherein in the step (8), reaction temperature of the amidation reactionis 25 degrees Celsius and reaction time is 48 hours.
 14. The method asclaimed in claim 10, wherein the amide is hydrolyzed to form a compoundDODGA whose structural formula is:


15. The method as claimed in claim 2, wherein the galactoside is6′-aminohexyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactoside,ah-GalNAc₄, having a structural formula of:


16. The method as claimed in claim 15, wherein the step of synthesizingthe galatosides further includes steps of step (9) using 6′-aminohexanoland benzyl chlorocarbonate to carry out an amino protecting reaction andget a 6′-(N-Benzyloxycarbonyl)aminohexanol; step (10) takingN-acetyl-D-galactosamine and acetyl chloride to react at 10° C. and get2-Acetamido-2,4,6,-tri-O-acetyl-1-chloro-1,2-dideoxy-α-D-galactopyranose;step (11) using 6′-(N-Benzyloxycarbonyl)aminohexanol and2-Acetamido-2,4,6,-tri-O-acetyl-1-chloro-1,2-dideoxy-α-D-galactopyranoseto perform substitution reaction in a mixture solution of toluene andnitromethane with a catalyst of mercuric cyanide and get6′-(N-Benzyloxycarbonyl)aminohexyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranoside.step (12) hydrogenizing/reducing 6′-(N-Benzyloxycarbonyl)aminohexyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosidein alcohol and in the presence of a palladium carbon catalyst to get the6′-aminohexyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactoside.17. The method as claimed in claim 2, wherein in the step of using thegalactosides to amidate the polycarboxyl groups of the amide, the amideis hydrolyzed to form a compound DODGA and the galactoside is6′-aminohexyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactoside,ah-GalNAc₄; the step of using the galactosides to amidate thepolycarboxyl groups of the amide further includes the steps of: step(13) activating a carboxylic acid of DODGA to react with ah-GalNAc₄ andusing 1,3-dicyclohexylcarbodiimide as well as N-hydroxysuccinimide as areagent to carry out the amidation reaction in chloroform solution andget6-Tri-o-(2′-acetamido-3′,4′,6′-tri-o-acetyl-2′-deoxy-β-D-galactopyranoside)hexyl7,10-diaza-9-oxo-7-[2-(triphenylmethyl)thioethyl]-12-[(triphenylmethyl)thio]-dodecanamido-γ-L-glutamyl-L-glutamate;and step (14) hydrolyzing6-Tri-o-(2′-acetamido-3′,4′,6′-tri-o-acetyl-2′-deoxy-β-D-galactopyranoside)hexyl7,10-diaza-9-oxo-7-[2-(triphenylmethyl)thioethyl]-12-[(triphenylmethyl)thio]-dodecanamido-γ-L-glutamyl-L-glutamatewith sodium methylate to get6-Tri-o-(2′-acetamido-3′,4′,6′-trihydroxy-2′-deoxy-β-D-galactopyranoside)hexyl7,10-diaza-9-oxo-7-[2-(triphenylmethyl)thioethyl]-12-[(triphenylmethyl)thio]-dodecanamido-γ-L-glutamyl-L-glutamate,which is (ah-GalNAc)₃-DODGA.
 18. A radiotracer of a precursor used forlabeling hepatocyte receptor and containing trisaccharide and a diamidedimercaptide (N₂S₂) ligand is represented by the following structuralformula:

wherein M is selected from the group consisting of ^(99m)Tc, ¹⁸⁸Re and¹¹¹In.
 19. A pharmaceutical composition of a precursor used for labelinghepatocyte receptor and containing trisaccharide and a diamidedimercaptide (N₂S₂) ligand is used to treat liver cancers and isrepresented by the following structural formula:

wherein M is selected from the group consisting of ^(99m)Tc, ¹⁸⁸Re and¹¹¹In.